problem in protein concentration

Hi
First if ur protein is eukaryotic in orgin, its a waste trying to get a correctly refolded protein from e.coli. If ur ptn is expressed in e.coli, try expressing the ptn in soluble form by varying with various temperature and harvesting time. Optimise a faverouble condition for getting the ptn in soluble form at least in little amounts. After down streaming processing u can concentrate the protein to the conc u need.
Why u have went for a elution buffer with both imidazole and urea? Its better to go for imidazole buffer ,if u carry ptn purification in native conditions.
Its always been hard to remove the urea w/o precipitating the protein. U can try dialysing by sequencially decreasing the urea conc in the dialysis buffer (6M-5M-4M...).
Find the theoritical PI of the protein and maintain the pH of the buffer away from it.
Gud luck

As far as knowing if the protein is refolded correctly, the easiest way is by some sort of functional assay. For example, a kinase should be able to phosphorylate its expected repertoire of targets if it has been refolded correctly.

The conditions for properly refolding a protein are largely dependent upon the particular protein in question. For this reason, I would recommend a kit, such as the one from Pierce (Protein Refolding Kit. This kit contains a variety of buffers and reagents for 9 different solubilization and refolding conditions. Also, the webpage contains sufficient information that you could essentially duplicate the kit by obtaining the constituents separately. AthenaES also has a refolding kit.

I have a protein fragment, about 31.6 kDA (with Theoretical pI between 6.05-6.15), with histidine tags which has been purified using Talon (more or less). As my protein formed inclusion bodies, I used 6M urea, meaning my protein is in an elution buffer containing 6M urea, 150mM imidazole, 50mM NaH2PO4 and 300mM NaCl (pH 7). Everytime I try to dialyse Urea out it starts to precipitate. I need the protein to at 1mg/ml concentration in buffer containing 50mM NaCl or less.
I really need this before I can continue with the rest of my project. I will appreciate any help/suggestions.
My main questions are
How can I prevent it from precipitating;
What buffer should I use and what pH should the buffer be at to keep my protein from precipitating;
Once I dialyse out the urea, how do I make sure that the protein refolded correctly
Thank you in advance. If you require more information let me know or email me any suggestions
snag769@gmail.com

Hi,

Its a well known that urea interferes ptn estimation, Try SDS gel to compare the dialysed sample with pre-dialysed sample. Hw come will u do ptn folding with urea & PEG? Remove the urea step by step to prevent ptn precipitation.

Mewryn

Thank you ........... amicon filters have molecular weight cut off so when we centrifuge our sample i have a doubt that whether 8M urea which has low molecular weight will go down that results in a precipitation of protein

The smallest MWCO you can get is 3 kDa. As long as your protein is bigger than this, it will be retained by the filter and the urea will pass through. The concentration of urea will remain at 8 M.

Thank you ........... amicon filters have molecular weight cut off so when we centrifuge our sample i have a doubt that whether 8M urea which has low molecular weight will go down that results in a precipitation of protein

Hi,
I am facing problem in concentrating my protein. I have my protein with 8M urea and it is dilute. I have to concentrate my protein (MW 47kDa) before refolding. I kept my protein sample in dialysis bag (MWCo 3kDa ) in PEG 20,000. It absorbed water and my sample volume got reduced . But when i estimated my sample using bradford reagent my protein concentration was less. I am sure that my protein couldn't come out of that dialysis bag. i doubted whether urea concentration increases and affected my protein during concentrating or for sample with 8M urea will bradford reagent gives good estimation? pls help me ...........
Thank U in advance

8M urea will most likely interfere with this assay. Some references give 3M as a cut-off for not interfering. You can always test it by running a blank with just 8M urea.

The method you used for concentrating could result in a concentration of urea greater than 8M. If you need the protein to remain in 8M urea, I would recommend an Amicon Ultra centrifugal concentrator, sold by Millipore link. They have very low protein binding and will maintain the concentrated protein in the starting buffer. You can also use these devices for buffer exchange, if you want to.

Hi,
I am facing problem in concentrating my protein. I have my protein with 8M urea and it is dilute. I have to concentrate my protein (MW 47kDa) before refolding. I kept my protein sample in dialysis bag (MWCo 3kDa ) in PEG 20,000. It absorbed water and my sample volume got reduced . But when i estimated my sample using bradford reagent my protein concentration was less. I am sure that my protein couldn't come out of that dialysis bag. i doubted whether urea concentration increases and affected my protein during concentrating or for sample with 8M urea will bradford reagent gives good estimation? pls help me ...........
Thank U in advance